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BBA - General Subjects (v.1726, #3)

Editorial Board (pp. ii).

Gain-of-function screen identifies a role of the Sec61α translocon in Drosophila postmitotic neurotoxicity by Hirotaka Kanuka; Tetsuo Hiratou; Tatsushi Igaki; Hiroshi Kanda; Erina Kuranaga; Kazunobu Sawamoto; Toshiro Aigaki; Hideyuki Okano; Masayuki Miura (pp. 225-237).
To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61α (DSec61α), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61α caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61α's translocon activity. This supported our previous observation that the DSec61α translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.

Keywords: Neurotoxicity; Cell death; Apoptosis; Neurodegeneration; Drosophila


Role of peptide primary sequence in polyphenol–protein recognition: An example with neurotensin by T. Richard; X. Vitrac; J.M. Merillon; J.P. Monti (pp. 238-243).
Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol–protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta- O-galloyl-d-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.

Keywords: Neurotensin; Polyphenol; NMR


Phylogeny of anion exchangers: Could trout AE1 conductive properties be shared by other members of the gene family? by Hélène Guizouarn; Richard Christen; Franck Borgese (pp. 244-250).
A phylogenetic tree of anion exchangers (AE) was performed in order to better understand relationships between the different known AE and how they arose. Indeed, the different known AE1 from mammals or fish do not exhibit the same transport features: all studied anion exchangers 1 (AE1) catalyse an electroneutral Cl−/HCO3− exchange through the plasma membrane; however, trout AE1 (tAE1) is able to spontaneously form an anion conductive pathway permeable to some inorganic cations (Na+ and K+) as well as to organic osmolytes such as taurine. Therefore, it has been proposed that this major erythrocyte membrane protein could play a key role for the cell volume regulation of trout red cells. By analogy, it was envisioned that other fish anion exchangers could play a similar role in osmolyte loss induced by erythrocyte swelling. We have cloned AE1 from Raja erinacea and Danio rerio and studied their properties after expression in Xenopus laevis oocytes. In this study, we show that none of them is able to induce any conductive pathway or taurine permeability in Xenopus oocytes. Our phylogenetic analyses show that, first, all present AE1 genes have a common ancestor distinct from that of AE2 and AE3 and second, tAE1 is a true AE1 ortholog. The question of whether tAE1 conductive properties are a derived character in the trout lineage within Euteleostei or whether other AE1 members can share these properties is then discussed.

Keywords: Anion exchanger; Band3; Phylogeny; Anion channel; Taurine; Erythrocyte


cDNA cloning and functional expression of KM+, the mannose-binding lectin from Artocarpus integrifolia seeds by Luis L.P. daSilva; Jeanne Blanco de Molfetta-Machado; Ademilson Panunto-Castelo; Jurgen Denecke; Gustavo Henrique Goldman; Maria-Cristina Roque-Barreira; Maria Helena S. Goldman (pp. 251-260).
KM+, a mannose-binding lectin present in the seeds of Artocarpus integrifolia, has interesting biological properties and potential pharmaceutical use [A. Panunto-Castelo, M.A. Souza, M.C. Roque-Barreira, J.S. Silva, KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection, Glycobiology 11 (2001) 1035–1042. ; L.L.P. daSilva, A. Panunto-Castelo, M.H.S. Goldman, M.C. Roque-Barreira, R.S. Oliveira, M.D. Baruffi, J.B. Molfetta-Machado, Composition for preventing or treating appearance of epithelia wounds such as skin and corneal wounds or for immunomodulating, comprises lectin, Patent number WO20041008. ]. Here, we have isolated clones encoding the full-length KM+ primary sequence from a cDNA library, through matrix PCR-based screening methodology. Analysis of KM+ nucleotide and deduced amino acid sequences provided strong evidence that it neither enters the secretory pathway nor undergoes post-translational modifications, which is in sharp contrast with jacalin, the more abundant lectin from A. integrifolia seeds. Current investigations into the KM+ properties are often impaired by the difficulty in obtaining sufficient quantities of jacalin-free KM+ through direct seed extraction. To obtain active recombinant protein (rKM+) in larger amounts, we tested three different expression systems. Expression vectors were constructed to produce: (a) rKM+ in E. coli in its native form, (b) rKM+ with GST as an N-terminal tag and (c) native rKM+ in Saccharomyces cerevisiae. The presence of the GST-tag significantly improved the overall rKM+ yield; however, most of the obtained rGST-KM+ was insoluble. Production of rKM+ in the yeast host yielded the highest quantities of soluble lectin that retained the typical high-mannose oligosaccharide-binding properties of the natural protein. The possible biotechnological applications of recombinant KM+ are discussed.

Keywords: Abbreviations; rKM+; recombinant KM+ produced in a heterologous system; jfKM+; KM+ extracted from jackfruit seeds; HRP; horseradish peroxidase glycoprotein; JRLs; jacalin-related lectins; CRD; carbohydrate recognition domainKM+ or artocarpin; Jackfruit lectin; cDNA cloning; Heterologous expression; Mannose-binding


Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space inhibits cell spreading by Ryoichi Yamaji; Emi Chatani; Naoki Harada; Kenji Sugimoto; Hiroshi Inui; Yoshihisa Nakano (pp. 261-271).
The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not α-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.

Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Extracellular space; Cell spreading; Cell morphology; Active center


Characteristics of transxylosylation by β-xylosidase from Aspergillus awamori K4 by Masahiro Kurakake; Tomiko Fujii; Masahiro Yata; Takumi Okazaki; Toshiaki Komaki (pp. 272-279).
The Aspergillus awamori K4 β-xylosidase gene ( Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger β-xylosidase gene with only five amino acid differences. K4 β-xylosidase had the same catalytic mechanism as family 3 β-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 β-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)- O-β-d-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-β-d-xylopyranoside, 6S- O-β-d-xylosyl maltitol (S: sorbitol residue) and 6G- O-β-d-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl–enzyme intermediate.

Keywords: β-xylosidase; Transglycosylation; Aspergillus; Glucosidase; Xylooligosaccharide


Human protein tau represses DNA replication in vitro by Wen Li; Xing Sheng Wang; Mei Hua Qu; Ying Liu; Rong Qiao He (pp. 280-286).
Here, in the experiments of both PCR and real-time PCR, a repression of DNA amplification was observed in the presence of protein tau. Furthermore, a strong repression appeared when an in vitro DNA replication assay was performed at the physiological temperature (37 °C). The incorporation of dNTP was markedly decreased to ∼12% of control by the presence of tau23 and to ∼15% by tau40. In the competitive experiments, the PCR product could be restored when the competitor DNA was added, indicating that the association of tau with the template gave rise to the repression. However, tau did not repress the yield of RNA in transcription, suggesting that tau was replaced or ejected from the template by the elongating T7 RNA polymerase.

Keywords: Abbreviations; dsDNA; double stranded DNA; T7 RNAP; T7 RNA polymeraseProtein tau; DNA replication; Transcription; Repression; PCR


Dispersion of meso-tetrakis( N-methylpyridinium-4-yl)porphyrin on [d(A-T) n]2 oligonucleotides by Taegi Park; Jong Moon Kim; Sung Wook Han; Dong-Jin Lee; Seog K. Kim (pp. 287-292).
The binding modes of the free-base meso-tetrakis( N-methylpyridinium-4-yl)porphyrin (TMPyP) complexed with [d(AT) n]2 oligonucleotides (where n=3–8, corresponding to 6 to 16 AT base pairs) were studied by circular dichroism (CD). When associated with the shortest oligonucleotide, ([d(AT)3]2), a bisignate CD spectrum was produced in the Soret absorption region at the mixing ratio between 2.0 and 0.25, corresponding to one TMPyP per 0.5 to 4 oligonucleotides. Apparent bisignate CD was attributed to a stacked TMPyP along the DNA. On the other hand, when the oligonucleotide length reaches one helical turn or longer, ([d(AT) n]2, n=6,7,8), TMPyP exhibited a positive CD signal, that corresponds to the monomeric groove binding mode, at the mixing ratio below 1.0 (one TMPyP per oligonucleotide). As the mixing ratio increases, the CD signal was best accounted for by the sum of the stacked and groove-binding TMPyP. At the intermediate oligonucleotide length ([d(AT) n]2, n=4,5), the CD spectrum appeared to be the sum of the stacked and groove binding TMPyP at all mixing ratios. Therefore, it is conclusive that the full dispersion of TMPyP requires at least one helical turn of the AT sequence at a mixing ratio below 1.0. Further increase of the mixing ratio resulted in the stacking of TMPyP even at the long oligonucleotides.

Keywords: Porphyrin; DNA; Oligonucleotide; Circular dichroism; Linear dichroism


Thermodynamic basis for antibody binding to Z-DNA: Comparison of a monoclonal antibody and its recombinant derivatives by Edmar Vaz de Andrade; Sonia Maria Freitas; Manuel Mateus Ventura; Andréa Queiroz Maranhão; Marcelo Macedo Brigido (pp. 293-301).
Antibody engineering represents a promising area in biotechnology. Recombinant antibodies can be easily manipulated generating new ligand and effector activities that can be used as prototype magic bullets. On the other hand, an extensive knowledge of recombinant antibody binding and stability features are essential for an efficient substitution. In this study, we compared the stability and protein binding properties of two recombinant antibody fragments with their parental monoclonal antibody. The recombinant fragments were a monomeric scFv and a dimeric one, harboring human IgG1 CH2–CH3 domains. We have used fluorescence titration quenching to determine the thermodynamics of the interaction between an anti-Z-DNA monoclonal antibody and its recombinant antibody fragments with Z-DNA. All the antibody fragments seemed to bind DNA similarly, in peculiar two-affinity states. Enthalpy–entropy compensation was observed for both affinity states, but a marked entropy difference was observed for the monomeric scFv antibody fragment, mainly for the high affinity binding. In addition, we compared the stability of the dimeric antibody fragment and found differences favoring the monoclonal antibody. These differences seem to derive from the heterologous expression system used.

Keywords: Enthalpy–entropy compensation; Anti-DNA; Protein engineering; Recombinant antibody; Pichia pastoris


A comparative study of neuronal and inducible nitric oxide synthases: Generation of nitric oxide, superoxide, and hydrogen peroxide by John Weaver; Supatra Porasuphatana; Pei Tsai; Sovitj Pou; Linda J. Roman; Gerald M. Rosen (pp. 302-308).
Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (NO), superoxide (O2−), and hydrogen peroxide (H2O2). SinceNO has been implicated in many physiological processes, the importance of O2− and H2O2 in regulating cell signaling byNO cannot be overlooked. Before addressing these questions, we investigated the production ofNO, O2−, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux ofNO from each isozyme supports differential biological activity. We found that the initial rate and sustained production ofNO was considerably greater for NOS 2 as compared to NOS 1. In the absence ofl-arginine, however, NOS 1 generation of O2− and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production ofNO, O2−, and H2O2 may define the specific physiologic function of each isozyme.

Keywords: Neuronal nitric oxide synthase; Inducible nitric oxide synthase; Nitric oxide; Superoxide; Hydrogen peroxide; Cell signaling


Flavonoids purified from Rhus verniciflua Stokes actively inhibit cell growth and induce apoptosis in human osteosarcoma cells by Hyon-Seok Jang; Sung-Ho Kook; Young-Ok Son; Jong-Ghee Kim; Young-Mi Jeon; Yong-Suk Jang; Ki-Choon Choi; Ju Kim; Seong-Kyu Han; Kyung-Yeol Lee; Byung-Keon Park; Nam-Pyo Cho; Jeong-Chae Lee (pp. 309-316).
Many studies have suggested that dietary flavonoids are anticancer agents that induce the apoptosis of cancer cells. However, the effects of flavonoids on the induction of apoptosis in osteosarcoma cells are unclear. Previously, a flavonoid fraction, consisting mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, herein named RCMF (the RVS chloroform- methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS). This study evaluated the effects of RCMF on the proliferation and apoptosis using human osteosarcoma (HOS) cells. The mechanism of growth inhibition of the HOS cells by the flavonoid fraction, RCMF, was also assessed. The results demonstrated that RCMF exhibited sensitive growth inhibition and induced apoptosis in HOS cells. PARP cleavage was closely associated with the RCMF-induced apoptosis of the HOS cells. Furthermore, the activation of caspase 8 and Bax, the inhibition of Bcl-2 expression, and the release of cytochrome c are believed to be involved in the RCMF-mediated apoptosis. Collectively, these findings suggest that RCMF is an agent which may be capable of inducing sensitive growth inhibition and apoptosis in HOS cells.

Keywords: Abbreviations; HOS; human osteosarcoma cells; RCMF; RVS chloroform-methanol fraction; RVS; Rhus verniciflua; Stokes; MTT; 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide; TUNEL; Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling; MPT; Mitochondrial permeability transition; PI; Propidium iodide Rhus verniciflua; Stokes; Flavonoid; Human osteosarcoma cell; Growth inhibition; Apoptosis


Structural characteristics of a lipid peroxidation product, trans-2-nonenal, that favour inhibition of membrane-associated phosphotyrosine phosphatase activity by Ángel Hernández-Hernández; María N. Garabatos; Marina C. Rodríguez; María L. Vidal; Abel López-Revuelta; José I. Sánchez-Gallego; Marcial Llanillo; Jesús Sánchez-Yagüe (pp. 317-325).
Protein-tyrosine phosphatases (PTPs) are very susceptible to oxidation by reactive oxygen species (ROS), which induce the oxidation of catalytic cysteines, thereby inactivating these PTPs. PTPs are also inactivated by treatment with different aldehydes (such as trans-2-nonenal), produced after tissue damage by ROS. However, the molecular mechanisms behind such aldehyde-due inactivation remain unknown. Using commercially available compounds, we examined the structural characteristics of trans-2-nonenal that allow the inhibition of platelet membrane-associated PTP activity, as well as how these compounds affect the dynamics of SH–, CO– and NH2– protein groups on the membranes. PTP was effectively inhibited by physiological amounts of trans-2-nonenal (1–10 μM). Incubation with trans-2-nonene (10 μM) also decreased PTP activity, although to a lower extent. Treatment with nonyl aldehyde almost eliminated PTP inhibition. Decreases in protein thiols were visible after trans-2-nonenal and trans-2-nonene treatments. Both the latter compounds also increased protein carbonyls (although trans-2-nonenal was more effective) and decreased protein amino groups to an equal extent. Collectively, our data indicate that α,β unsaturation (and not a double bond in another position) is the most important structural determinant for PTP inhibition, the alkenal with 9-carbon atoms being the most effective in eliciting such inhibition. The data allow us to predict the modification of sulfhydryls and/or the formation of addition products with lysyl or histidyl residues, and hence the kind of specific antibodies that it would be necessary to generate in order to test such modifications directly.

Keywords: Abbreviations; BHA; 3(2)-tert-butyl-4-hydroxyanisole; DNS-Cl; dansyl chloride; DNPH; 2-4-dinitrophenylhydrazine; DTNB; 5,5′-dithiobis(2-nitrobenzoate); 4-HHE; 4-hydroxy-2-trans-hexenal; 4-HNE; 4-hydroxy-2-trans-nonenal; PTP; protein tyrosine phosphatase; PUFA; polyunsaturated fatty acids; ROS; reactive oxygen species; TCA; trichloroacetic acidTrans-2-nonenal; Protein tyrosine phosphatase; Lipid peroxidation; Platelet


Expression of tcaA and mprF and glycopeptide resistance in clinical glycopeptide-intermediate Staphylococcus aureus (GISA) and heteroGISA strains by M. Wootton; A.P. Macgowan; T.R. Walsh (pp. 326-327).
Two genes recently associated with glycopeptide intermediate resistance in Staphylococcus aureus (GISA) are mprF and tcaA, with inactivation causing shifts in vancomycin resistance. This study reveals that expression levels of both genes are similar in groups of clinical GISA, heteroGISA and glycopeptide susceptible strains, suggesting no association with clinical isolates.

Keywords: Glycopeptide-intermediate resistance; Staphylococcus aureus; Gene expression; Clinical isolate; Mechanism of resistance


Fucoxanthin induces cell cycle arrest at G0/G1 phase in human colon carcinoma cells through up-regulation of p21WAF1/Cip1 by Swadesh K. Das; Takashi Hashimoto; Kazuo Shimizu; Tatsushi Yoshida; Toshiyuki Sakai; Yoshihiro Sowa; Akitoshi Komoto; Kazuki Kanazawa (pp. 328-335).
Fucoxanthin, a natural carotenoid, has been reported to have antitumorigenic activity in mouse colon, skin and duodenum models. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against colon cancer using the human colon adenocarcinoma cell lines. Fucoxanthin reduced the viability of WiDr cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during the G0/G1 phase at 25 μM and apoptosis at 50 μM. Fucoxanthin at 25 μM inhibited the phosphorylation of the retinoblastoma protein (pRb) at Ser780 and Ser807/811 24 h after treatment without changes in the protein levels of the D-types of cyclin and cyclin-dependent kinase (cdk) 4, whose complexes are responsible for the phosphorylation of pRb at these sites. A cdk inhibitory protein, p21WAF1/Cip1 increased 24 h after the treatment with 25 μM of fucoxanthin, but not p27Kip1. In addition, the mRNA of p21WAF1/Cip1 also increased in a dose-dependent manner. According to the experiments using the isogenic human colon adenocarcinoma cell lines, fucoxanthin failed to induce G0/G1 arrest in the p21-deficient HCT116 cells, but not in HCT116 wild-type cells. All of these findings showed that fucoxanthin inhibited proliferation of colon cancer cells. The inhibitory mechanism is due to the cell cycle arrest during the G0/G1 phase mediated through the up-regulation of p21WAF1/Cip1, which may be related to the antitumorigenic activity.

Keywords: Fucoxanthin; p21; WAF/Cip1; Cell cycle arrest at G; 0; /G; 1; phase; Apoptosis; Colon carcinoma cell
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